Focused Ion Beam-scanning Electron Research Articles

Pt-Black-Modified (Hemi)spherical AFM Sensors: In Situ Imaging of Light-Driven Hydrogen Peroxide Evolution.

In this work, we present (hemi)spherical atomic force microscopy (AFM) sensors for the detection of hydrogen peroxide. Platinum-black (Pt-B) was electrodeposited onto conductive colloidal AFM probes or directly at recessed microelectrodes located at the end of a tipless cantilever, resulting in electrocatalytically active cantilever-based sensors that have a small geometric area but, due to the porosity of the films, exhibit a large electroactive surface area. Focused ion beam-scanning electron microscopy tomography revealed the porous 3D structure of the deposited Pt-B. Given the accurate positioning capability of AFM, these probes are suitable for local in situ sensing of hydrogen peroxide and at the same time can be used for (electrochemical) force spectroscopy measurements. Detection limits for hydrogen peroxide in the nanomolar range (LOD = 68 ± 7 nM) were obtained. Stability test and first in situ proof-of-principle experiments to achieve the electrochemical imaging of hydrogen peroxide generated at a microelectrode and at photocatalytically active structured poly(heptazine imide) films are demonstrated. Force spectroscopic data of the photocatalyst films were recorded in ambient conditions, in solution, and by applying a potential, which demonstrates the versatility of these novel Pt-B-modified spherical AFM probes.

Multiscale investigation of pore structure heterogeneity in carbonate rocks using digital imaging and SCAL measurements: A case study from Upper Jurassic limestones, Abu Dhabi, UAE.

This study presents a comprehensive analysis of rock properties for a selected group of six carbonate reservoir rock samples revealing complex structures at various length scales. Experimental laboratory methods as well as image analysis techniques were conducted in this study to characterize the macro- and micro-pores in mud- and grain-dominated limestones samples from the Upper Jurassic Arab Formation (Arab D member). Mercury Injection Capillary Pressure (MICP), porosimeter, and permeameter lab measurements were employed to assess the pore network heterogeneity and complexity. In addition, a multiscale rock imaging approach was implemented to detect grain and pore phases at several length scales using Thin Sections (TS), Scanning Electron Microscopy (SEM), Focused Ion Beam Scanning Electron Microscopy (FIB-SEM), as well as 3D X-ray Computed Tomography (CT), and micro-computed tomography images (MCT). Furthermore, the multifractal analysis method was applied on the MICP and FIB-SEM to characterize quantitatively the heterogeneity of the pores in the studied samples. Heterogeneous samples 3R, 4M, 5W, and 6M display the highest non-uniformity degree Δα values, falling within the range of [1.21, 1.39] based on FIB-SEM images. Samples 1G, 2R, 3R, and 5W exhibit more heterogeneous pore structure, with Δα values ranging from 0.73 to 1.49 based on the MICP experiments. The results and findings confirm the effectiveness of multifractal parameters Δα and the asymmetry degree in the vertical axis Δf(α) in quantifying and characterizing rock heterogeneity.

Three-dimensional correlative microscopy of the Drosophila female reproductive tract reveals modes of communication in seminal receptacle sperm storage

In many taxa, females store sperm in specialized storage organs. Most insect sperm storage organs have a tubular structure, typically consisting of a central lumen surrounded by epithelial cells. These specialized tubules perform the essential tasks of transporting sperm through the female reproductive tract and supporting long-term sperm survival and function. Little is known about the way in which female sperm storage organs provide an environment conducive to sperm survival. We address this using a combined light microscopy, micro computed tomography (microCT), and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) approach for high-resolution correlative three-dimensional imaging to advance our understanding of sperm-female interactions in Drosophila melanogaster. Using this multimodal approach, we were able to scan the lower female reproductive tract and distal portion of the seminal receptacle at low magnification, and to subsequently zoom in for further analysis on an ultrastructural level. Our findings highlight aspects of the way in which the seminal receptacle keeps sperm viable in the lumen, and set the stage for further studies. The methods developed are suitable not only for Drosophila but also for other organisms with soft, delicate tissues.

Quantitative characterization of organic and inorganic pores in shale based on deep learning

Organic matter (OM) maturity is closely related to organic pores in shales. Quantitative characterization of organic and inorganic pores in shale is crucial for rock-physics modeling and reservoir porosity and permeability evaluation. Focused ion beam-scanning electron microscopy (FIB-SEM) can capture high-precision three-dimensional (3D) images and directly describe the types, shapes, and spatial distribution of pores in shale gas reservoirs. However, due to the high scanning cost, wide 3D view field, and complex microstructure of FIB-SEM, more efficient segmentation for the FIB-SEM images is required. For this purpose, a multiphase segmentation workflow in conjunction with a U-net is developed to segment pores from the matrix and distinguish organic pores from inorganic pores simultaneously in the entire 3D image stack. The workflow is repeated for FIB-SEM data sets of 17 organic-rich shales with various characteristics. The analysis focuses on improving the efficiency and relevance of the workflow, that is, quantifying the minimum number of training slices while ensuring accuracy and further combining the fractal dimension (FD) and lacunarity to study a simple and objective method of selection. Meanwhile, the computational efficiency, accuracy, and robustness to noise of the 2D U-net model are discussed. The intersection over the union of automatic segmentation can amount to 80%–95% in all data sets with manual labels as ground truth. In addition, calculated by the FIB-SEM multiphase segmentation, the organic porosity is used to quantitatively evaluate the OM decomposition level. Deep-learning-based segmentation shows great potential for characterizing shale pore structures and quantifying OM maturity.

Focused ion beam-SEM 3D study of osteodentin in the teeth of the Atlantic wolfish Anarhichas lupus

The palette of mineralized tissues in fish is wide, and this is particularly apparent in fish dentin. While the teeth of all vertebrates except fish contain a single dentinal tissue type, called orthodentin, dentin in the teeth of fish can be one of several different tissue types. The most common dentin type in fish is orthodentin. Orthodentin is characterized by several key structural features that are fundamentally different from those of bone and from those of osteodentin. Osteodentin, the second-most common dentin type in fish (based on the tiny fraction of fish species out of ∼30,000 extant fish species in which tooth structure was so far studied), is found in most Selachians (sharks and rays) as well as in several teleost species, and is structurally different from orthodentin.Here we examine the hypothesis that osteodentin is similar to anosteocytic bone tissue in terms of its micro- and nano-structure. We use Focused Ion Beam-Scanning Electron Microscopy (FIB/SEM), as well as several other high-resolution imaging techniques, to characterize the 3D architecture of the three main components of osteodentin (denteons, inter-denteonal matrix, and the transition zone between them). We show that the matrix of osteodentin, although acellular, is extremely similar to mammalian osteonal bone matrix, both in general morphology and in the three-dimensional nano-arrangement of its mineralized collagen fibrils. We also document the presence of a complex network of nano-channels, similar to such networks recently described in bone. Finally, we document the presence of strings of hyper-mineralized small ‘pearls’ which surround the denteonal canals, and characterize their structure.

A Dynamic Permeability Model in Shale Matrix after Hydraulic Fracturing: Considering Mineral and Pore Size Distribution, Dynamic Gas Entrapment and Variation in Poromechanics

Traditional research on apparent permeability in shale reservoirs has mainly focussed on effects such as poromechanics and porosity-assisted adsorption layers. However, for a more realistic representation of field conditions, a comprehensive multi-scale and multi-flowing mechanism model, considering the fracturing process, has not been thoroughly explored. To address this research gap, this study introduces an innovative workflow for dynamic permeability assessment. Initially, an accurate description of the pore size distribution (PSD) within three major mineral types in shale is developed using focussed ion beam-scanning electron microscopy (FIB-SEM) and nuclear magnetic resonance (NMR) data. Subsequently, an apparent permeability model is established by combining the PSD data, leading to the derivation of dynamic permeability. Finally, the PSD-related dynamic permeability model is refined by incorporating the effects of imbibition resulting from the fracturing process preceding shale gas production. The developed dynamic permeability model varies with pore and fracture pressures in the shale reservoir. The fracturing process induces water blockage, water-film formation, and water-bridging phenomena in shale, requiring additional pressure inputs to counteract capillary effects in hydrophilic minerals in shale, But also increases the overall permeability from increasing permeability at larger scale pores. Unlike traditional reservoirs, the production process commences when the fracture is depleted to 1–2 MPa exceeds the pore pressure, facilitated by the high concentration of hydrophobic organic matter pores in shale, this phenomenon explains the gas production at the intial production stage. The reduction in adsorption-layer thickness resulting from fracturing impacts permeability on a nano-scale by diminishing surface diffusion and the corresponding slip flow of gas. this phenomenon increases viscous-flow permeability from enlarged flow spacing, but the increased viscous flow does not fully offset the reduction caused by adsorbed-gas diffusion and slip flow. In addition to the phenomena arising from various field conditions, PSD in shale emerges as a crucial factor in determining dynamic permeability. Furthermore, considering the same PSD in shale, under identical pore spacing, the shape factor of slit-like clay minerals significantly influences overall permeability characteristics, much more slit-shaped pores(higher shape factor) reduce the overall permeability. The dynamic permeability-assisted embedded discrete fracture model (EDFM) showed higher accuracy in predicting shale gas production compared to the original model.

Evaluation of ER;CR:YSGG Laser and Remineralization Agents on Mineral Density and Ion Levels of Primary and Permanent Enamel.

Objective: The aim of this study is to evaluate the efficacy of two different fluoride varnishes used alone or in combination with laser treatment on permanent and primary tooth enamel. Methods: Ninety-six primary and 96 permanent molar samples were divided into six groups. The levels of calcium, phosphorus, fluoride, and silver ions of each sample were analyzed using energy-dispersive X-ray spectroscopy (EDS). Six different treatments were applied to 12 different groups (n = 15) as control (g1/G1), fluoride varnish (g2/G2), casein phosphopeptide-amorphous calcium phosphate (CPP-ACP)-fluoride varnish (g3/G3), laser (g4/G4), laser+fluoride varnish (g5/G5), and laser+CPP-ACP-fluoride varnish (g6/G6). After the procedures, ion levels were reanalyzed with EDS. The teeth were subjected to the artificial caries-forming procedure and ion levels were again evaluated by EDS. One sample from each group was prepared separately for the focused ion beam-scanning electron microscope measurement; initial and final images were recorded. The obtained data were statistically analyzed with the SPSS 23.0 program. Results: Compared with the initial measurement, phosphorus percentages increased in most of the groups in the last measurement. Calcium percentages of primary teeth increased in the last measurement, except for the g1 group, but in permanent teeth, there was an increase only in the G6 group. There was a statistically significant difference between g1/G1 and g6/G6 groups in the last measurement of phosphorus and calcium percentages. Conclusions: The combined use of laser with CPP-ACP-fluoride varnish enhanced remineralization in the primary and permanent teeth. However, in permanent teeth, the use of laser alone was not as effective as in primary teeth. Therefore, combined usage with CPP-ACP-fluoride varnish can enhance its efficacy. This in vitro study was approved by the local ethics committee of Hacettepe University (Project No.: GO 20/441).

The Mg Electrode Cycling Mechanism in Simple Salt Glyme Electrolytes

The lithium-ion battery (LIB) has been at the forefront of innovations in energy storage since its commercialisation over 30 years ago. As we reach the practical specific capacity limit of current state-of-the-art materials, novel and innovative battery technologies are required to satiate the requirements of energy intensive applications such as electric vehicles. 1 The use of a lithium metal negative electrode has been proposed to increase the energy density, however the high reactivity causes poor cycling performance due to electrolyte degradation, dendrite formation and safety issues. 2 The magnesium electrode offers a solution that has double the theoretical volumetric capacity of the lithium electrode while simultaneously mitigating dendritic growth, reducing raw material costs and greatly increasing sustainability. 3,4 Previous work has shown that traditional LIB electrolytes are not compatible with magnesium metal, and the plating and stripping mechanism that underpins how magnesium electrodes cycle is poorly understood. 5 Here we explored the mechanism of magnesium plating and stripping in glyme-based electrolytes with a range of additional additives. We show that in the pure electrolyte solution, interphase formation is critical to stable cycling but that this is accompanied by significant degradation and accumulation of inactive Mg.6 We establish the three-dimensional chemical composition of Mg deposits in the different electrolyte formulations using a combination of focused ion beam-scanning electron microscopy (FIB-SEM), energy dispersive x-ray spectroscopy (EDX) and transmission electron microscopy (TEM). We use these findings to develop a mechanistic understanding that explains the electrochemical cycling performance of magnesium metal negative electrodes. In situ electrochemical quartz crystal microbalance (EQCM) measurements are used to evaluate the plating and stripping efficiency of the electrodes and to identify non-electrochemical degradation reactions between Mg and the electrolyte solutions.

MUSCLE UP: WHAT AGING LOOKS LIKE FROM THE INSIDE OUT

Abstract The Longitudinal Studies Section of the Translational Gerontology Branch of the National Institute on Aging Intramural Research Program has the mission and opportunity to examine aging physiology from the cellular to the full body functional level in model systems and human populations. This symposium presents ongoing research that targets pathologic and normative change in muscle with aging and the significance of these changes for understanding the aging process. Data come from the Baltimore Longitudinal Study of Aging (BLSA) and the Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing (GESTALT) study. In nearly 500 BLSA participants aged 50+, Zenobia Moore evaluates the association between multiple epigenetic age metrics and change over 6 years in mid-femur areas of muscle and subcutaneous and intramuscular fat derived from computed tomography. Qu (Teresa) Tian investigates the relationship between skeletal muscle mitochondrial function and risk of MCI or dementia over 3.3 years in 460 initially cognitively normal BLSA participants and PET and blood biomarkers of AD in available subsamples. In 82 men and women from GESTALT, Stefano Donega examines relationships between physical activity, peak oxygen consumption and muscle oxidative capacity using p31 MR spectroscopy and gene expression, transcript usage and splicing events derived from vastus lateralis biopsies. Using volumetric Focused Ion Beam Scanning Electron Microscopy, Lisa Hartnell shares 3D images of muscle samples at the level of the mitochondrial reticulum and sarcomere from young and old healthy and frail GESTALT participants. Together, these findings highlight the value of muscle as a window on aging.

Segmentation of cellular ultrastructures on sparsely labeled 3D electron microscopy images using deep learning.

Focused ion beam-scanning electron microscopy (FIB-SEM) images can provide a detailed view of the cellular ultrastructure of tumor cells. A deeper understanding of their organization and interactions can shed light on cancer mechanisms and progression. However, the bottleneck in the analysis is the delineation of the cellular structures to enable quantitative measurements and analysis. We mitigated this limitation using deep learning to segment cells and subcellular ultrastructure in 3D FIB-SEM images of tumor biopsies obtained from patients with metastatic breast and pancreatic cancers. The ultrastructures, such as nuclei, nucleoli, mitochondria, endosomes, and lysosomes, are relatively better defined than their surroundings and can be segmented with high accuracy using a neural network trained with sparse manual labels. Cell segmentation, on the other hand, is much more challenging due to the lack of clear boundaries separating cells in the tissue. We adopted a multi-pronged approach combining detection, boundary propagation, and tracking for cell segmentation. Specifically, a neural network was employed to detect the intracellular space; optical flow was used to propagate cell boundaries across the z-stack from the nearest ground truth image in order to facilitate the separation of individual cells; finally, the filopodium-like protrusions were tracked to the main cells by calculating the intersection over union measure for all regions detected in consecutive images along z-stack and connecting regions with maximum overlap. The proposed cell segmentation methodology resulted in an average Dice score of 0.93. For nuclei, nucleoli, and mitochondria, the segmentation achieved Dice scores of 0.99, 0.98, and 0.86, respectively. The segmentation of FIB-SEM images will enable interpretative rendering and provide quantitative image features to be associated with relevant clinical variables.

Bundling of collagen fibrils influences osteocyte network formation during bone modeling

Osteocytes form a cellular network by gap junctions between their cell processes. This network is important since intercellular communication via the network is essential for bone metabolism. However, the factors that influence the formation of this osteocyte network remain unknown. As the early stage of osteocyte network formation occurs on the bone surface, we observed a newly formed trabecular bone surface by orthogonal focused ion beam-scanning electron microscopy. The embedding late osteoblast processes tended to avoid bundled collagen fibrils and elongate into sparse collagen fibrils. Then, we examined whether the inhibition of bundling of collagen fibrils using a potent lysyl oxidase inhibitor, β-aminopropionitrile (BAPN) changed the cellular network of the chick calvaria. The osteocyte shape of the control group was spindle-shape, while that of the BAPN group was sphere-shaped. In addition, the osteocyte processes of the control group were elongated vertically to the long axis of the cell body, whereas the osteocyte processes of the BAPN group were elongated radially. Therefore, it was suggested that the bundling of collagen fibrils influences normal osteocyte network formation during bone modeling.

Characterization for 3D pore-fracture systems in Hot-Dry-Rock samples from Gonghe Basin, China

As a new type of renewable energy, Hot-Dry-Rock (HDR) is gaining increasing attention. The physical properties of rock influenced by the pore-fracture structure are vital for the hydraulic and chemical characteristics of HDR reservoirs. However, the natural pore-fracture structure in HDR has not been sufficiently studied. To fulfill this gap, we used focused ion beam-scanning electron microscopy (FIB-SEM) to construct three-dimensional (3D) digital models for four granite samples from the deep HDR reservoir in Gonghe Basin, Qinghai Province, China, and obtained the nanoscale structure of the pores and fractures in HDR reservoir for the first time. Our analysis based on the 3D models indicates that the heat transfer area in the HDR reservoir is mainly from fractures and crack-like pores, but a large amount of small but regular pores could be neglected when heat transfer in HDR is considered. The permeability of HDR reservoirs at nanoscale varies from 9.414 nD to 0.082 mD, and the high permeability in this study is accompanied by throughgoing fractures. The variation of porosity and permeability of deep HDR at nanoscale in this study is much more severe than that of shallow granites previously studied at microscale. The performance of geothermal field is significantly influenced by the size of throats and the average size and tortuosity of the interconnected pore-fracture network in HDR. The hydraulic aperture could be 32-58% lower than the mechanical aperture of the natural fractures in HDR reservoirs, which is a vital clue for the numerical simulation associated with enhanced geothermal systems.

Visualizing the Structure, Composition and Activity of Single Catalyst Particles for Olefin Polymerization and Polyolefin Decomposition.

The structural and morphological characterization of individual catalyst particles for olefin polymerization, as well as for the reverse process of polyolefin decomposition, can provide an improved understanding for how these catalyst materials operate under relevant reaction conditions. In this review, we discuss an emerging analytical toolbox of 2D and 3D chemical imaging techniques that is suitable for investigating the chemistry and reactivity of related catalyst systems. While synchrotron-based X-ray microscopy still provides unparalleled spatial resolutions in 2D and 3D, a number of laboratory-based techniques, most notably focused ion beam-scanning electron microscopy, confocal fluorescence microscopy, infrared photoinduced force microscopy and laboratory-based X-ray nano-computed tomography, have helped to significantly expand the arsenal of analytical tools available to scientists in heterogeneous catalysis and polymer science. In terms of future research, the review outlines the role and impact of in situ and operando (spectro-)microscopy experiments, involving sophisticated reactors as well as online reactant and product analysis, to obtain real-time information on the formation, decomposition, and mobility of polymer phases within single catalyst particles. Furthermore, the potential of fluorescence microscopy, X-ray microscopy and optical microscopy is highlighted for the high-throughput characterization of olefin polymerization and polyolefin decomposition catalysts. By combining these chemical imaging techniques with, for example, chemical staining methodologies, selective probe molecules as well as particle sorting approaches, representative structure-activity relationships can be derived at the level of single catalyst particles.

Use of Heat-killed Aspergillus ochraceus NRRL 3174 Discs as Biosorbent for petroleum Removal

The purpose of this work was to evaluate the petroleum sorption capacity of heat-killed fungal discs obtained from Aspergillus ochraceus strain. Effect of various parameters such as biosorbent dose (0.5g-2.5g/100mL), petroleum concentration (0.5-5%), pH (4.0-8.0), contact time (1-12h) and re-usability of biosorbent (1-6) were investigated. Accordingly, the highest biosorption capacity was obtained with 1% petroleum concentration, 1.5 g/100mL heat-killed fungal discs, 10h contact time at pH: 5.0 and room temperature. Additionally, each disc was able to actively use for at least 6 more cycles in biosorption experiments. The specific removal rate was calculated as 0.114 day−1, the rate constant and half-life period were also 1.609 day-1, t1/2 = 0.431, respectively. The kinetic study was described by the pseudo-second order model and the equilibrium modeling was found to be well fitted with Langmuir isotherm. The biosorbent(s) were characterized by Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). Over 80% removal of long-chain n-alkanes by the heat-killed fungal discs was confirmed by GC-MS analysis. Since there has been no similar study investigating the sorption of petroleum with heat-killed Aspergillus ochraceous discs, this novel bio-based sorbent with its low cost, environmentally friendly and easy-to-apply properties can be used in advanced biosorption studies.

Revealing the aging process of solid electrolyte interphase on SiOx anode

As one of the most promising alternatives to graphite negative electrodes, silicon oxide (SiOx) has been hindered by its fast capacity fading. Solid electrolyte interphase (SEI) aging on silicon SiOx has been recognized as the most critical yet least understood facet. Herein, leveraging 3D focused ion beam-scanning electron microscopy (FIB-SEM) tomographic imaging, we reveal an exceptionally characteristic SEI microstructure with an incompact inner region and a dense outer region, which overturns the prevailing belief that SEIs are homogeneous structure and reveals the SEI evolution process. Through combining nanoprobe and electron energy loss spectroscopy (EELS), it is also discovered that the electronic conductivity of thick SEI relies on the percolation network within composed of conductive agents (e.g., carbon black particles), which are embedded into the SEI upon its growth. Therefore, the free growth of SEI will gradually attenuate this electron percolation network, thereby causing capacity decay of SiOx. Based on these findings, a proof-of-concept strategy is adopted to mechanically restrict the SEI growth via applying a confining layer on top of the electrode. Through shedding light on the fundamental understanding of SEI aging for SiOx anodes, this work could potentially inspire viable improving strategies in the future.

Porosity evolution in proton irradiated microfine-grained POCO graphite

This work investigated the porosity evolution of POCO ZXF-5Q graphite that has been irradiated by 340 kW, 120 GeV protons inside NT02 target system in Fermilab's NuMI beamline. This POCO graphite has undergone direct bulk dimensional swelling at low dose irradiation and its local microstructural change is still not well-understood during this process. In this work, the (sub-) micrometre scale porosity from six locations across proton beam fluence and temperature gradients have been studied using focused ion beam-scanning electron microscopy (FIB-SEM) tomography. A deep learning-based tomographic image segmentation technique has been established and implemented for porosity segmentation and quantification. It has been found that there is a decrease in the total volumetric percentage of the porosity at proton beam centre (∼ 8 – 8.4 vol.%), by comparing to un-irradiated POCO (∼ 12 – 13 vol.%) and to beam 2σ and 5σ radii (∼ 12 vol.%). This decrease in porosity volume percentage was found to be caused by the reduction in pores with volumes > 0.1 μm3 induced by material bulk dimensional swelling at proton beam centre area. The porosity reduction in relation to dimensional change and irradiation creep was discussed among with other contributing factors, and further investigations through well-controlled irradiation experiment are still needed.

Three dimensional microstructures of carbon deposition on Ni-YSZ anodes under polarization

In the present study, two Ni/YSZ anodes with different volume ratios of Ni and YSZ, 30: 70 and 45: 55 vol%, are operated in dry methane under open circuit and polarized conditions. Three-dimensional (3D) Ni/YSZ microstructures after carbon deposition are reconstructed by the focused ion beam-scanning electron microscopy (FIB-SEM) with the help of machine learning segmentation. From the reconstructed mircostructures, volume fraction, connectivity, three phase boundary (TPB) density, and tortuosity are quantified. In addition, local carbon microstructures are quantitatively reconstructed, and the effect of polarization on carbon morphology is investigated. It is demonstrated that Ni surface in the vicinity of active TPB near the electrolyte is free from carbon formation, while remaining Ni surface at some distances from TPB exhibits severe carbon deposition. In average, total amount of carbon deposition is larger near the electrolyte. These observations imply complex interplay between the electrochemical steam generation and methane cracking on Ni surface which take place very locally near the active TPB.

3D relationship between hierarchical canal network and gradient mineralization of shark tooth osteodentin.

Osteodentin is a dominant mineralized collagenous tissue in the teeth of many fishes, with structural and histological characteristics resembling those of bone. Osteodentin, like bone, comprises osteons as basic structural building blocks, however, it lacks the osteocytes and the lacuno-canalicular network (LCN), which are known to play critical roles in controlling the mineralization of the collagenous matrix in bone. Although numerous vascular canals exist in osteodentin, their role in tooth maturation and the matrix mineralization process remain poorly understood. Here, high resolution micro-computed tomography (micro-CT) and focused ion beam-scanning electron microscopy (FIB-SEM) were used to obtain 3D structural information of osteodentin in shark teeth at multiple scales. We observed a complex 3D network of primary canals with a diameter ranging from ∼10 µm to ∼120 µm, where the canals are surrounded by osteon-like concentric layers of lamella, with 'interosteonal' tissue intervening between neighboring osteons. In addition, numerous hierarchically branched secondary canals extended out radially from the primary canals into the interosteonal tissue, with a decreasing diameter from ∼10 µm to hundreds of nanometers. Interestingly, the mineralization degree increases from the periphery of primary canals to the interosteonal tissue, suggesting that mineralization begins in the interosteonal tissue. Correspondingly, the hardness and elastic modulus of the interosteonal tissue is higher than those of the osteonal tissue. These results demonstrate that the 3D hierarchical canal network is positioned to play a critical role in controlling the gradient mineralization of osteodentin, also providing valuable insight into the formation of mineralized collagenous tissue without osteocytes and LCN. STATEMENT OF SIGNIFICANCE: : Bone is a composite material with versatile mechanical properties. Osteocytes and lacuno-canalicular network (LCN) are kown to play critical roles during formation of human bone. However, the skeleton of many fishes, including bone and osteodentin, although lacking osteocytes and LCN, could exhibit similar osteon-like structure and mechanical functions. Here, using various high resolution 3D characterization techniques, we reveal that the 3D network of primary canals and numerous hierarchically branched secondary canals correlate with the mineralization gradient and micromechanical properties of osteonal and interosteonal tissues of shark tooth osteodentin. This work significantly improves our understanding of the construction of bone-like mineralized tissue without osteocytes and LCN, and provides inspirations for the fabrication of functional materials with hierarchical structure.

An interactive deep learning-based approach reveals mitochondrial cristae topologies.

The convolution of membranes called cristae is a critical structural and functional feature of mitochondria. Crista structure is highly diverse between different cell types, reflecting their role in metabolic adaptation. However, their precise three-dimensional (3D) arrangement requires volumetric analysis of serial electron microscopy and has therefore been limiting for unbiased quantitative assessment. Here, we developed a novel, publicly available, deep learning (DL)-based image analysis platform called Python-based human-in-the-loop workflow (PHILOW) implemented with a human-in-the-loop (HITL) algorithm. Analysis of dense, large, and isotropic volumes of focused ion beam-scanning electron microscopy (FIB-SEM) using PHILOW reveals the complex 3D nanostructure of both inner and outer mitochondrial membranes and provides deep, quantitative, structural features of cristae in a large number of individual mitochondria. This nanometer-scale analysis in micrometer-scale cellular contexts uncovers fundamental parameters of cristae, such as total surface area, orientation, tubular/lamellar cristae ratio, and crista junction density in individual mitochondria. Unbiased clustering analysis of our structural data unraveled a new function for the dynamin-related GTPase Optic Atrophy 1 (OPA1) in regulating the balance between lamellar versus tubular cristae subdomains.

The fate of mitochondria during platelet activation

AbstractBlood platelets undergo several successive motor-driven reorganizations of the cytoskeleton when they are recruited to an injured part of a vessel. These reorganizations take place during the platelet activation phase, the spreading process on the injured vessel or between fibrin fibers of the forming clot, and during clot retraction. All these steps require a lot of energy, especially the retraction of the clot when platelets develop strong forces similar to those of muscle cells. Platelets can produce energy through glycolysis and mitochondrial respiration. However, although resting platelets have only 5 to 8 individual mitochondria, they produce adenosine triphosphate predominantly via oxidative phosphorylation. Activated, spread platelets show an increase in size compared with resting platelets, and the question arises as to where the few mitochondria are located in these larger platelets. Using expansion microscopy, we show that the number of mitochondria per platelet is increased in spread platelets. Live imaging and focused ion beam–scanning electron microscopy suggest that a mitochondrial fission event takes place during platelet activation. Fission is Drp1 dependent because Drp1-deficient platelets have fused mitochondria. In nucleated cells, mitochondrial fission is associated with a shift to a glycolytic phenotype, and using clot retraction assays, we show that platelets have a more glycolytic energy production during clot retraction and that Drp1-deficient platelets show a defect in clot retraction.